RESUMO
Live-attenuated influenza vaccines (LAIV) offer advantages over the commonly used inactivated split influenza vaccines. However, finding the optimal balance between sufficient attenuation and immunogenicity has remained a challenge. We recently developed an alternative LAIV based on the 2009 pandemic H1N1 virus with a truncated NS1 protein and lacking PA-X protein expression (NS1(1-126)-ΔPAX). This virus showed a blunted replication and elicited a strong innate immune response. In the present study, we evaluated the efficacy of this vaccine candidate in the porcine animal model as a pertinent in vivo system. Immunization of pigs via the nasal route with the novel NS1(1-126)-ΔPAX LAIV did not cause disease and elicited a strong mucosal immune response that completely blocked replication of the homologous challenge virus in the respiratory tract. However, we observed prolonged shedding of our vaccine candidate from the upper respiratory tract. To improve LAIV safety, we developed a novel prime/boost vaccination strategy combining primary intramuscular immunization with a haemagglutinin-encoding propagation-defective vesicular stomatitis virus (VSV) replicon, followed by a secondary immunization with the NS1(1-126)-ΔPAX LAIV via the nasal route. This two-step immunization procedure significantly reduced LAIV shedding, increased the production of specific serum IgG, neutralizing antibodies, and Th1 memory cells, and resulted in sterilizing immunity against homologous virus challenge. In conclusion, our novel intramuscular prime/intranasal boost regimen interferes with virus shedding and transmission, a feature that will help combat influenza epidemics and pandemics.
Assuntos
Administração Intranasal , Vacinas contra Influenza , Infecções por Orthomyxoviridae , Animais , Suínos , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/imunologia , Injeções Intramusculares , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/administração & dosagem , Vírus da Influenza A Subtipo H1N1/imunologia , Modelos Animais de Doenças , Anticorpos Antivirais/imunologia , Imunização Secundária/métodos , Vacinação/métodos , Influenza Humana/prevenção & controle , Influenza Humana/imunologiaRESUMO
Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.
Assuntos
Quirópteros/virologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Especificidade de Hospedeiro , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Zoonoses/imunologia , Zoonoses/virologia , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Galinhas/genética , Galinhas/imunologia , Quirópteros/genética , Quirópteros/imunologia , Quirópteros/metabolismo , Feminino , Perfilação da Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Especificidade de Hospedeiro/genética , Especificidade de Hospedeiro/imunologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Sistema Respiratório/virologia , Suínos/genética , Suínos/imunologia , Tropismo Viral/genética , Tropismo Viral/imunologia , Replicação Viral , Zoonoses/genética , Zoonoses/metabolismoRESUMO
The influenza A virus (IAV) M2 protein has proton channel activity, which plays a role in virus uncoating and may help to preserve the metastable conformation of the IAV hemagglutinin (HA). In contrast to the highly conserved M2 proteins of conventional IAV, the primary sequences of bat IAV H17N10 and H18N11 M2 proteins show remarkable divergence, suggesting that these proteins may differ in their biological function. We, therefore, assessed the proton channel activity of bat IAV M2 proteins and investigated its role in virus replication. Here, we show that the M2 proteins of bat IAV did not fully protect acid-sensitive HA of classical IAV from low pH-induced conformational change, indicating low proton channel activity. Interestingly, the N31S substitution not only rendered bat IAV M2 proteins sensitive to inhibition by amantadine but also preserved the metastable conformation of acid-sensitive HA to a greater extent. In contrast, the acid-stable HA of H18N11 did not rely on such support by M2 protein. When mutant M2(N31S) protein was expressed in the context of chimeric H18N11/H5N1(6:2) encoding HA and NA of avian IAV H5N1, amantadine significantly inhibited virus entry, suggesting that ion channel activity supported virus uncoating. Finally, the cytoplasmic domain of the H18N11 M2 protein mediated rapid internalization of the protein from the plasma membrane leading to low-level expression at the cell surface. However, cell surface levels of H18N11 M2 protein were significantly enhanced in cells infected with the chimeric H18N11/H5N1(6:2) virus. The potential role of the N1 sialidase in arresting M2 internalization is discussed. IMPORTANCE Bat IAV M2 proteins not only differ from the homologous proteins of classical IAV by their divergent primary sequence but are also unable to preserve the metastable conformation of acid-sensitive HA, indicating low proton channel activity. This unusual feature may help to avoid M2-mediated cytotoxic effects and inflammation in bats infected with H17N10 or H18N11. Unlike classical M2 proteins, bat IAV M2 proteins with the N31S substitution mediated increased protection of HA from acid-induced conformational change. This remarkable gain of function may help to understand how single point mutations can modulate proton channel activity. In addition, the cytoplasmic domain was found to be responsible for the low cell surface expression level of bat IAV M2 proteins. Given that the M2 cytoplasmic domain of conventional IAV is well known to participate in virus assembly at the plasma membrane, this atypical feature might have consequences for bat IAV budding and egress.
Assuntos
Quirópteros , Vírus da Influenza A , Animais , Amantadina/farmacologia , Linhagem Celular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/fisiologia , Virus da Influenza A Subtipo H5N1/metabolismo , PrótonsRESUMO
Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged-Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.
Assuntos
Autofagia/genética , Sistemas CRISPR-Cas , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , SARS-CoV-2/genética , Antivirais/farmacologia , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Replicação ViralRESUMO
To investigate the class-dependent properties of anti-viral IgM antibodies, we use membrane antigen capture activated cell sorting to isolate spike-protein-specific B cells from donors recently infected with SARS-CoV-2, allowing production of recombinant antibodies. We isolate 20, spike-protein-specific antibodies of classes IgM, IgG, and IgA, none of which shows any antigen-independent binding to human cells. Two antibodies of class IgM mediate virus neutralization at picomolar concentrations, but this potency is lost following artificial switch to IgG. Although, as expected, the IgG versions of the antibodies appear to have lower avidity than their IgM parents, this is not sufficient to explain the loss of potency.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Monoclonais , Anticorpos Antivirais , Humanos , Imunoglobulina G , Imunoglobulina MRESUMO
The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.
Assuntos
Tratamento Farmacológico da COVID-19 , Anticorpos de Domínio Único , Anticorpos Neutralizantes , Anticorpos Antivirais/metabolismo , Resistência a Medicamentos , Humanos , Pandemias , Ligação Proteica , SARS-CoV-2/genética , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/farmacologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Bats are natural reservoirs of numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction between coronaviruses and bat cells is sparse. We investigated the ability of primary cells from Rhinolophus and Myotis species, as well as of established and novel cell lines from Myotis myotis, Eptesicus serotinus, Tadarida brasiliensis, and Nyctalus noctula, to support SARS-CoV-2 replication. None of these cells were permissive to infection, not even the ones expressing detectable levels of angiotensin-converting enzyme 2 (ACE2), which serves as the viral receptor in many mammalian species. The resistance to infection was overcome by expression of human ACE2 (hACE2) in three cell lines, suggesting that the restriction to viral replication was due to a low expression of bat ACE2 (bACE2) or the absence of bACE2 binding in these cells. Infectious virions were produced but not released from hACE2-transduced M. myotis brain cells. E. serotinus brain cells and M. myotis nasal epithelial cells expressing hACE2 efficiently controlled viral replication, which correlated with a potent interferon response. Our data highlight the existence of species-specific and cell-specific molecular barriers to viral replication in bat cells. These novel chiropteran cellular models are valuable tools to investigate the evolutionary relationships between bats and coronaviruses. IMPORTANCE Bats are host ancestors of several viruses that cause serious disease in humans, as illustrated by the ongoing SARS-CoV-2 pandemic. Progress in investigating bat-virus interactions has been hampered by a limited number of available bat cellular models. We have generated primary cells and cell lines from several bat species that are relevant for coronavirus research. The various permissivities of the cells to SARS-CoV-2 infection offered the opportunity to uncover some species-specific molecular restrictions to viral replication. All bat cells exhibited a potent entry-dependent restriction. Once this block was overcome by overexpression of human ACE2, which serves at the viral receptor, two bat cell lines controlled well viral replication, which correlated with the inability of the virus to counteract antiviral responses. Other cells potently inhibited viral release. Our novel bat cellular models contribute to a better understanding of the molecular interplays between bat cells and viruses.
Assuntos
Quirópteros , SARS-CoV-2 , Replicação Viral , Enzima de Conversão de Angiotensina 2/genética , Animais , Quirópteros/virologia , Humanos , Receptores Virais/metabolismo , SARS-CoV-2/fisiologia , Especificidade da Espécie , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
The C-type lectin receptor DC-SIGN is a pattern recognition receptor expressed on macrophages and dendritic cells. It has been identified as a promiscuous entry receptor for many pathogens, including epidemic and pandemic viruses such as SARS-CoV-2, Ebola virus, and HIV-1. In the context of the recent SARS-CoV-2 pandemic, DC-SIGN-mediated virus dissemination and stimulation of innate immune responses has been implicated as a potential factor in the development of severe COVID-19. Inhibition of virus binding to DC-SIGN, thus, represents an attractive host-directed strategy to attenuate overshooting innate immune responses and prevent the progression of the disease. In this study, we report on the discovery of a new class of potent glycomimetic DC-SIGN antagonists from a focused library of triazole-based mannose analogues. Structure-based optimization of an initial screening hit yielded a glycomimetic ligand with a more than 100-fold improved binding affinity compared to methyl α-d-mannopyranoside. Analysis of binding thermodynamics revealed an enthalpy-driven improvement of binding affinity that was enabled by hydrophobic interactions with a loop region adjacent to the binding site and displacement of a conserved water molecule. The identified ligand was employed for the synthesis of multivalent glycopolymers that were able to inhibit SARS-CoV-2 spike glycoprotein binding to DC-SIGN-expressing cells, as well as DC-SIGN-mediated trans-infection of ACE2+ cells by SARS-CoV-2 spike protein-expressing viruses, in nanomolar concentrations. The identified glycomimetic ligands reported here open promising perspectives for the development of highly potent and fully selective DC-SIGN-targeted therapeutics for a broad spectrum of viral infections.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Moléculas de Adesão Celular/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Superfície Celular/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismoRESUMO
Arenaviruses are a large family of emerging enveloped negative-strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human-pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of "druggable" targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the antiarenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel antiarenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole derivatives for therapeutic intervention against human-pathogenic arenaviruses.IMPORTANCE Emerging human-pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel antiarenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotrimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo Our study provides the basis for further evaluation of clotrimazole derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate repurposing for therapeutic intervention against human-pathogenic arenaviruses.
Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Clotrimazol/farmacologia , Fusão de Membrana/efeitos dos fármacos , Células A549 , Animais , Infecções por Arenaviridae/tratamento farmacológico , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Endocitose/efeitos dos fármacos , Células HEK293 , Células HeLa , Febres Hemorrágicas Virais/tratamento farmacológico , Febres Hemorrágicas Virais/virologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Vírus Lassa/efeitos dos fármacos , Células Vero , Proteínas do Envelope Viral/metabolismo , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacosRESUMO
Immune responses induced by currently licensed inactivated influenza vaccines are mainly directed against the hemagglutinin (HA) glycoprotein, the immunodominant antigen of influenza viruses. The resulting antigenic drift of HA requires frequent updating of the vaccine composition and annual revaccination. On the other hand, the levels of antibodies directed against the neuraminidase (NA) glycoprotein, the second major influenza virus antigen, vary greatly. To investigate the potential of the more conserved NA protein for the induction of subtype-specific protection, vesicular stomatitis virus-based replicons expressing a panel of N1 proteins from prototypic seasonal and pandemic H1N1 strains and human H5N1 and H7N9 isolates were generated. Immunization of mice and ferrets with the replicon carrying the matched N1 protein resulted in robust humoral and cellular immune responses and protected against challenge with the homologous influenza virus with an efficacy similar to that of the matched HA protein, illustrating the potential of the NA protein as a vaccine antigen. The extent of protection after immunization with mismatched N1 proteins correlated with the level of cross-reactive neuraminidase-inhibiting antibody titers. Passive serum transfer experiments in mice confirmed that these functional antibodies determine subtype-specific cross-protection. Our findings illustrate the potential of NA-specific immunity for achieving broader protection against antigenic drift variants or newly emerging viruses carrying the same NA but a different HA subtype.IMPORTANCE Despite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths worldwide. Currently licensed inactivated vaccines, which are standardized for the amount of the hemagglutinin (HA) antigen, primarily induce strain-specific antibodies, whereas the immune response to the neuraminidase (NA) antigen, which is also present on the viral surface, is usually low. Using NA-expressing single-cycle vesicular stomatitis virus replicons, we show that the NA antigen conferred protection of mice and ferrets against not only the matched influenza virus strains but also viruses carrying NA proteins from other strains of the same subtype. The extent of protection correlated with the level of cross-reactive NA-inhibiting antibodies. This highlights the potential of the NA antigen for the development of more broadly protective influenza vaccines. Such vaccines may also provide partial protection against newly emerging strains with the same NA but a different HA subtype.
Assuntos
Proteção Cruzada/imunologia , Neuraminidase/antagonistas & inibidores , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Reações Cruzadas , Furões , Hemaglutininas/imunologia , Humanos , Imunidade Celular , Imunização Passiva , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Neuraminidase/classificação , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Replicon , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
Two novel influenza A-like viral genome sequences have recently been identified in Central and South American fruit bats and provisionally designated "HL17NL10" and "HL18NL11." All efforts to isolate infectious virus from bats or to generate these viruses by reverse genetics have failed to date. Recombinant vesicular stomatitis virus (VSV) encoding the hemagglutinin-like envelope glycoproteins HL17 or HL18 in place of the VSV glycoprotein were generated to identify cell lines that are susceptible to bat influenza A-like virus entry. More than 30 cell lines derived from various species were screened but only a few cell lines were found to be susceptible, including Madin-Darby canine kidney type II (MDCK II) cells. The identification of cell lines susceptible to VSV chimeras allowed us to recover recombinant HL17NL10 and HL18NL11 viruses from synthetic DNA. Both influenza A-like viruses established a productive infection in MDCK II cells; however, HL18NL11 replicated more efficiently than HL17NL10 in this cell line. Unlike conventional influenza A viruses, bat influenza A-like viruses started the infection preferentially at the basolateral membrane of polarized MDCK II cells; however, similar to conventional influenza A viruses, bat influenza A-like viruses were released primarily from the apical site. The ability of HL18NL11 or HL17NL10 viruses to infect canine and human cells might reflect a zoonotic potential of these recently identified bat viruses.
RESUMO
Vesicular stomatitis virus (VSV) expressing the Ebola virus (EBOV) glycoprotein (GP) in place of the VSV glycoprotein G (VSV/EBOV-GP) is a promising EBOV vaccine candidate which has already entered clinical phase 3 studies. Although this chimeric virus was tolerated overall by volunteers, it still caused viremia and adverse effects such as fever and arthritis, suggesting that it might not be sufficiently attenuated. In this study, the VSV/EBOV-GP vector was further modified in order to achieve attenuation while maintaining immunogenicity. All recombinant VSV constructs were propagated on VSV G protein expressing helper cells and used to immunize guinea pigs via the intramuscular route. The humoral immune response was analysed by EBOV-GP-specific fluorescence-linked immunosorbent assay, plaque reduction neutralization test and in vitro virus-spreading inhibition test that employed recombinant VSV/EBOV-GP expressing either green fluorescent protein or secreted Nano luciferase. Most modified vector constructs induced lower levels of protective antibodies than the parental VSV/EBOV-GP or a recombinant modified vaccinia virus Ankara vector encoding full-length EBOV-GP. However, the VSV/EBOV-GP(F88A) mutant was at least as immunogenic as the parental vaccine virus although it was highly propagation-restricted. This finding suggests that VSV-vectored vaccines need not be propagation-competent to induce a robust humoral immune response. However, VSV/EBOV-GP(F88A) rapidly reverted to a fully propagation-competent virus indicating that a single-point mutation is not sufficient to maintain the propagation-restricted phenotype.
Assuntos
Ebolavirus/imunologia , Glicoproteínas/imunologia , Imunogenicidade da Vacina , Vesiculovirus/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais , Ebolavirus/genética , Vetores Genéticos , Glicoproteínas/genética , Cobaias , Imunidade Humoral , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Vacinação , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genéticaRESUMO
Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned Mx1 cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat Mx1 genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat Mx1 genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts.IMPORTANCE Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity against Ebola virus and bat influenza A-like virus, and we describe here their phylogenetic relationship, revealing patterns of positive selection that suggest a coevolution with viral pathogens. By understanding the molecular mechanisms of the innate resistance of bats against viral diseases, we might gain important insights into how to prevent and fight human zoonotic infections caused by bat-borne viruses.
Assuntos
Antivirais/metabolismo , Quirópteros/imunologia , Quirópteros/virologia , Proteínas de Resistência a Myxovirus/metabolismo , Vírus de RNA/imunologia , Animais , Clonagem Molecular , Evolução Molecular , Proteínas de Resistência a Myxovirus/genética , Seleção GenéticaRESUMO
There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transient-expression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP- and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLP-generating MVA recombinant.IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) is a safe and immunogenic vaccine vector with a large capacity to accommodate multiple foreign genes. In this study, we combined the advantages of VLPs and the MVA platform by generating a recombinant MVA-BN-EBOV-VLP that would produce noninfectious EBOV VLPs in the vaccinated individual. Our results show that human cells infected with MVA-BN-EBOV-VLP indeed formed and released EBOV VLPs, thus producing a highly authentic immunogen. MVA-BN-EBOV-VLP efficiently induced EBOV-specific humoral and cellular immune responses in vaccinated mice. These results are the basis for future advancements, e.g., by including antigens from various filoviral species to develop multivalent VLP-producing MVA-based filovirus vaccines.
Assuntos
Vacinas contra Ebola/imunologia , Ebolavirus/isolamento & purificação , Glicoproteínas/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vaccinia virus/genética , Vírion/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD8-Positivos/imunologia , Vacinas contra Ebola/genética , Ebolavirus/genética , Ebolavirus/imunologia , Ebolavirus/fisiologia , Glicoproteínas/genética , Humanos , Imunoglobulina G/sangue , Camundongos , Nucleoproteínas/genética , Nucleoproteínas/imunologia , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Vírion/fisiologiaAssuntos
Vacinas contra COVID-19 , COVID-19 , Feminino , Masculino , Humanos , SARS-CoV-2 , COVID-19/prevenção & controle , Vacinação , ImunidadeRESUMO
In 2012 and 2013, influenza virus genome sequences of two new influenza A virus (IAV) subtypes were discovered in bat specimens, but further characterization was largely impeded by the lack of infectious virus. With the identification of highly susceptible cell lines, reconstitution of infectious bat IAV by reverse genetics recently succeeded and allowed a first insight into the life cycle of these viruses. Although there is a certain degree of functional compatibility between bat and conventional influenza A virus proteins, there are striking differences, including receptor usage, polarity of infection and reassortment potential.
Assuntos
Quirópteros/virologia , Vírus da Influenza A/genética , Infecções por Orthomyxoviridae/veterinária , Animais , Infecções por Orthomyxoviridae/virologiaAssuntos
Imunidade Humoral , Falência Renal Crônica , Anticorpos Antivirais , Humanos , Imunidade Celular , VacinaçãoRESUMO
UNLABELLED: The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain that is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably as a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, the antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in an NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activities of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spread of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high-quality NA antibodies for controlling influenza virus replication and place emphasis on NA as a vaccine antigen. IMPORTANCE: The neuraminidase of influenza A viruses is a sialidase that acts as a receptor-destroying enzyme facilitating the release of progeny virus from infected cells. Here, we demonstrate that monospecific anti-NA immune sera inhibited not only sialidase activity, but also influenza virus hemagglutination and infection of MDCK cells, suggesting that NA antibodies can interfere with virus attachment. Inhibition of both processes, virus release and virus binding, may explain why NA antibodies efficiently blocked virus dissemination in vitro and in vivo. Anti-NA immune sera showed broader reactivity than anti-HA sera in hemagglutination inhibition tests and demonstrated cross-subtype activity in sialidase inhibition tests. These remarkable features of NA antibodies highlight the importance of the NA antigen for the development of next-generation influenza virus vaccines.
Assuntos
Soros Imunes/imunologia , Vírus da Influenza A/imunologia , Neuraminidase/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Galinhas , Cães , Influenza Aviária/prevenção & controle , Neuraminidase/administração & dosagem , Suínos , Proteínas Virais/administração & dosagem , Internalização do Vírus , Liberação de Vírus/imunologia , Eliminação de Partículas ViraisRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.
Assuntos
Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Replicon/imunologia , Vacinas Virais/imunologia , Viremia/veterinária , Animais , Glicoproteínas/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Suínos , Vacinas Sintéticas/imunologia , Vesiculovirus/genética , Vesiculovirus/imunologia , Proteínas Virais/metabolismo , Viremia/imunologia , Viremia/prevenção & controle , Vírion/imunologiaRESUMO
Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.